1. Field of the Invention
The present invention relates generally to the fields of biology and medicine. More particularly, it concerns a process for the ex vivo formation of bone and uses thereof.
2. Description of Related Art
The development of a functional tissue such as bone requires the concerted action of a number of microenvironmental signals: cytokines/growth factors, extracellular matrix (ECM) molecules, and cell:cell interactions. Moreover, these regulatory signals must be queued in the appropriate temporal and spatial order, resulting in a developmental microenvironment that facilitates three-dimensional growth. The skeletal system is no exception to such requirements. It is well understood that a number of cytokines/growth factors, such as TGF-β1 family members, modulate bone formation, and that ECM molecules like osteonectin, osteocalcin, and Type I and II collagen, etc., are important in both osteogenesis and chondrogenesis. As opposed to in vitro systems that are predominately planar, the need for three-dimensional tissue-like development is implicit both in the structural nature of the skeleton and its embryonic development. However, the extension of these in vivo spatial requirements to in vitro systems has been difficult and largely overlooked.
Cellular condensation, a process of cell aggregation mediated by mesenchymal:epithelial cell interactions, plays a crucial role during skeletogenesis (Hall and Miyake, 1992; 1995; Stringa et al., 1997). In the developing chick embryo, cellular condensation precedes differentiation into to prechondrocytes (Hall and Miyake, 1995). In contrast, during osteogenesis, cells differentiate to preosteoblasts and then undergo condensation (Hall and Miyake, 1995; Centrella, 1987). This condensation nonetheless precedes osteoblast differentiation and matrix mineralization (Dunlop and Hall, 1995). Studies of prechondrocytes demonstrate that cell condensation is cytokine-mediated, and induces changes in the expression of a number of developmentally important genes. For example, TGF-β1 or BMP2 both stimulate chondrocytic condensation and up-regulate fibronectin, N-CAM, and tenascin (Hall and Miyake, 1995). The requisite step of cellular condensation during mesenchymal chondrogenesis is mimicked in vitro in chondrocyte micromass cultures (Denker et al., 1995). Tuan and colleagues have demonstrated that TGF-β1 treatment of the multipotent C3H10T1/2 cells in small volumes of media at high cell density (i.e., micro-mass cultures) results in the formation of three dimensional structures that are cartilaginous in nature (Denker et al., 1995). These cellular condensations are associated with the up-regulation of cartilage extracellular matrix components such as Type II collagen and cartilage link protein (Denker et al., 1995). Likewise, studies of embryonic chick (calverial or limb-bud) cells confirm the cell-density mediated induction of chondrogenesis (Wong and Tuan, 1995; Woodward and Tuan, 1999), and demonstrate an obligate requirement for cell:cell interaction in this process, most likely mediated by N-cadherin (Woodward and Tuan, 1999), or N-CAM (Oberlender and Tuan, 1994; Miyake et al., 1996).
To date, no in vitro models of tissue-like osteogenic cell growth or cellular-condensation exist. Calverial or bone marrow-derived osteogenic cells are typically grown on two-dimensional (i.e., planar) surfaces. Cell proliferation eventually leads to a localized piling of confluent cells into “bone nodules.” This suggests that cell-density plays a role in the process of bone formation; however, studies directly demonstrating a relationship between cell-density and bone-formation are lacking, as are studies demonstrating the formation of three dimensional, crystalline bone (as opposed to reports concerning the mineralization of the extracellular matrix surrounding bone).
Present methods for the repair of bony defects include grafts of organic and synthetic construction. Three types of organic grafts are commonly used: autografts, allografts, and xenografts. An autograft is tissue transplanted from one site to another in the patient. The benefits of using the patient's own tissue is that the graft will not evoke an immune response. However, using an autograft requires a second surgical site, which increases the risk of infection and may introduce additional complications. Further, bone available for grafting comes from a limited number of sites, for example, the fibula, ribs and iliac crest. An allograft is tissue taken from a different organism of the same species, and a xenograft from an organism of a different species. The latter types of tissue are readily available in larger quantities than autografts, but genetic differences between the donor and recipient may lead to rejection of the graft. All have advantages and disadvantages, yet none provides a perfect replacement for the missing bone.
There exists a need for a better way to repair and/or replace bone in subjects suffering from bone diseases or bone traumas.